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心血管 研究 研讨会Stanford-ChinaCardiovascular

SymposiumSeptember 21-22, 2017

Li Ka Shing Center | Paul Berg Hall | Stanford, CA

Sponsored in partby the Chi-Li Pao Foundation

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8:30 am Registration & Continental Breakfast

9:00 am Opening RemarksJoseph C. Wu, MD, PhDDirector, Stanford Cardiovascular Institute Simon H. Stertzer Professor of Medicine and Radiology, Stanford

9:05 am Welcome from the School of MedicineLloyd B. Minor, MDCarl and Elizabeth Naumann ProfessorDean of Stanford University School of Medicine

9:10 am Welcome from Stanford Health CareDavid Entwistle, MHSAPresident and CEO of Stanford Health Care

9:15 am Welcome from Lucile Packard Children’s HospitalChristopher G. Dawes, MBAPresident and CEO of Lucile Packard Children’s Hospital

9:20 am Keynote

Introduction to Keynote Speakerby Simon H. Stertzer, MDProfessor of Medicine, Emeritus, Stanford

The Cardiovascular Disease Continuum: Nearly 30 Years Later Victor J. Dzau, MDPresident, National Academy of MedicineJames B. Duke Professor of Medicine, Duke University

CARDIAC SURGERYModerator: Anson Lee, MD Assistant Professor of Cardiothoracic Surgery Stanford University

9:50 am Convergence of Biologic and Surgical Reconstructive Therapiesfor Cardiovascular Diseases Y. Joseph Woo, MDNorman E. Shumway ProfessorChair, Department of Cardiothoracic Surgery, Stanford

SCHEDULE: Day 1

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10:00 am Congenital Heart Disease: Pulmonary Artery ReconstructionFrank Hanley, MDLawrence Crowley, MD, Endowed Professor in Child Health, Stanford

10:10 am Heart Failure and Heart Transplant Program in Beijing Anzhen HospitalHaibo Zhang, MD, PhDProfessor, Deputy Director, Department of Cardiac SurgeryBeijing Anzhen Hospital, Capital Medical University

10:20 am Robotic Cardiac Surgery in China: Experience from PLA 301 HospitalRong Wang, MD Associate Professor and Director, Department of Cardiovascular SurgeryChinese PLA General Hospital

10:30 am Panel discussion with “Cardiac Surgery” speakers

10:50 am Coffee Break

CLINICAL SCIENCEModerator: Stephen Roth, MDProfessor of Pediatrics (Cardiology)Lucile Packard Children’s Hospital

11:10 am Prenatal and Postpartum Integration Management in China Yihua He, MD, PhDProfessor and Chair, Consultation Center for Maternal-Fetal Medicine in Fetal Heart Disease, Beijing Anzhen Hospital No. 2

11:20 am Heart Rhythm Control: The Original Works of Fuwai

Yan Yao, MD, PhDVice Director, Arrhythmia Center of Fuwai HospitalPeking Union Medical College & Chinese Academy of Medical Sciences

11:30 am The Future of Arrhythmia Therapy Paul Wang, MD Professor of Medicine (Cardiovascular Medicine), StanfordEditor of ‘Circulation: Arrhythmia & Electrophysiology’

11:40 am Panel discussion with “Clinical Science” speakers

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12:00 p.m. Networking Lunch and Poster Viewing

1:00 pm Keynote

Introduction to Keynote Speakerby Li Wang, MD, PhDProfessor, State Key Laboratory of Cardiovascular Disease, Fuwai HospitalPeking Union Medical College & Chinese Academy of Medical Sciences

The Unceasing Battle against Cardiovascular Diseases in China: Fuwai’s Mission Now and Beyond

Zhe Zheng, MD, PhDProfessor, Deputy Director, National Center for Cardiovascular Diseases Vice President, Fuwai HospitalPeking Union Medical College & Chinese Academy of Medical Sciences

VASCULAR SURGERY/PERIPHERAL ARTERIAL DISEASE Moderator: Nicholas Leeper, MDAssociate Professor of Surgery, Vascular Surgery and Medicine Stanford University

1:25 pm Aortic Aneurysm PathogenesisRonald L. Dalman, MDChief, Division of Vascular SurgeryWalter Clifford Chidester and Elsa Rooney Chidester Professor of Surgery, Stanford

1:35 pm The Management of Aortic Arch Disease: Open, Hybrid or EndoChang Shu, MD, PhDProfessor & Director, Department of Vascular Surgery, Fuwai HospitalPeking Union Medical College & Chinese Academy of Medical Sciences

1:45 pm Present Situation of Chinese Treatment of Type A Acute Aortic Dissection/Overview of Anzhen

Ming Gong, MD, PhDAssociate Professor, Beijing Anzhen Hospital, Capital Medical University

1:55 pm Recent Advances in Small Diameter Blood Vessel RegenerationDeling Kong, PhDProfessor & Dean of College of Life Sciences, Nankai University

2:05 pm Panel discussion with “Vascular Surgery/Peripheral Arterial Disease” speakers

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2:25 pm Coffee Break

Basic ScienceModerator: Xin-Liang ‘Xin’ Ma, MD, PhDProfessor, Medicine and Emergency MedicineDirector, Cardiovascular Research Program, Thomas Jefferson University

2:35 pm Uncovering the Roles of miRNA in Cardiovascular Physiology and DiseasesDao Wen Wang, MD, PhDProfessor & Director, Department of Internal Medicine, Tongji HospitalHuazhong University of Science and Technology

2:45 pm Single Cell Profiling Identifies INO80 as a Critical Chromatin Remodeler Dictating Cardiac Lineage Commitment

Li Wang, MD, PhDProfessor, State Key Laboratory of Cardiovascular Disease, Fuwai HospitalPeking Union Medical College & Chinese Academy of Medical Sciences

2:55 pm Development of the Right Sided Heart and Cardiac RegenerationZhongzhou Yang, PhDProfessor, Model Animal Research Center, Nanjing University

3:05 pm Applying Systems Immunology Methods to Understand Cardiovascular Health and Disease

Mark M. Davis, PhD Director, Stanford Institute of Immunity, Transplantation, and InfectionThe Burt and Marion Avery Family Professor, Stanford

3:15 pm Panel discussion with “Basic Science” speakers

3:35 pm Break

Interventional CardiologyModerator: David Lee, MDAssociate Professor, Cardiovascular Medicine Stanford University

3:55 pm Transcatheter Valve Therapy: Current and Future Alan Yeung, MDLi Ka Shing Professor in CardiologyChief, Cardiovascular Medicine (Clinical), Stanford

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4:05 pm Can We Predict Myocardial Infarction: The Ongoing Search for the ‘Vulnerable Plaque’William Fearon, MDProfessor of Medicine, Cardiovascular MedicineDirector, Interventional Cardiology, Stanford

4:15 pm Interventional Therapy in Atherosclerosis Yida Tang, MD, PhDProfessor & Director, Department of Internal Medicine, Fuwai HospitalPeking Union Medical College & Chinese Academy of Medical Sciences

4:25 pm Panel discussion with “Interventional Cardiology” speakers

4:45 pm Introduction to the Chinese Edition of the NEJM (医学前沿)Rui-Ping Xiao, MD, PhDProfessor at the Institute of Molecular MedicinePeking University

4:55 pm Adjourn

8:30 am Continental Breakfast

8:55 am Introduction to Chi-Li Pao Foundation Programs Gloria Kim, MDClinical Assistant Professor, Medicine (Cardiovascular Medicine)Medical Director, International Medical Services, Stanford Health Care

9:00 am Keynote

Introduction to Keynote Speakerby Daniel Bernstein, MDAlfred Woodley Salter and Mabel Smith Salter Endowed Professor in Pediatrics, Stanford

Identification of Novel Allosteric Drugs for G Protein Coupled Receptors Brian Kobilka, MD2012 Nobel LaureateHelene Irwin Fagan Chair in Cardiology and Professor of Molecular & CellularPhysiology, Stanford

SCHEDULE: Day 2

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CLINICAL TRIALS, INNOVATION & PARTNERSHIPSModerator: Kenneth Mahaffey, MDProfessor of Medicine (Cardiovascular Medicine)Vice Chair of Clinical Research, Stanford

9:25 am Surgery for End-Stage Heart Failure: Practice and Experience in Fuwai Hospital Zhe Zheng, MD, PhDVice President, Fuwai HospitalPeking Union Medical College & Chinese Academy of SciencesProfessor & Deputy Director, National Center for Cardiovascular Diseases

9:35 am Atrial Fibrillation: Personalizing Care and Improving Quality Through Global Programs Mintu Turakhia, MD, MASAssociate Professor of Medicine, Cardiovascular Medicine and by courtesy, of Health and Research Policy, Veterans Affairs Health Care SystemSenior Director of Research and Innovation

9:45 am Academia–Industry Partnerships Robert A. Harrington, MDArthur L. Bloomfield Professor of MedicineChair Stanford Department of Medicine

9:55 am Panel discussion with “Clinical Trials, Innovation & Partnerships” speakers

10:15 Coffee Break

IMAGING Moderator: Phillip Yang, MDAssociate Professor of Medicine (Cardiovascular Medicine)Stanford University

10:25 am Clinical Need and Future of Cardiac Imaging Bin Lu, MD, PhDProfessor & Director, Department of Radiology, Fuwai HospitalPeking Union Medical College & Chinese Academy of Medical Sciences

10:35 am Comprehensive Cardiac CT Strategies for Coronary Disease Management Koen Nieman, MD, PhDAssociate Professor, Cardiovascular Medicine, Stanford

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10:45 am Multimodality Molecular Imaging of Progressive Atherosclerosis Feng Cao, MD, PhDProfessor & Deputy Director, Department of CardiologyChinese PLA General Hospital

10:55 am Panel discussion with “Imaging” speakers

11:15 am Networking Lunch and Poster Viewing

1:00 pm Keynote

Introduction of Keynote Speakerby Mark Mercola, PhD Professor, Cardiovascular Medicine, Stanford

Converged Cardiac Cell Death Signaling: A Tale of CaMKIIRui-Ping Xiao, MD, PhDProfessor at the Institute of Molecular Medicine, Peking University, Beijing, China

STEM CELLS & CARDIAC REGENERATIONModerator: Jianyi ‘Jay’ Zhang, MD, PhD Chair, Department of Biomedical Engineering, University of Alabama, Birmingham

1:30 pm Overview of Translational Stem Cell Research for Heart Failure at ZhejiangHeart Center Hong Yu, PhD, FAHAProfessor, Key Lab for Cardiovascular Diseases, The 2nd Affiliated HospitalZhejiang University School of Medicine

1:40 pm Progressing in Pluripotent Stem Cell-derived Cardiac Lineage CellsHuangtian Yang, PhDProfessor, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

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1:50 pm The Long and the Short of It: Telomere Lengths Predict Cardiac FailureHelen Blau, MDThe Donald E. and Delia B. Baxter Foundation Professor, Stanford

2:00 pm Critical Regulations of Niche on Stem Cell Fate and Cardiac RegenerationXi-Yong Yu, MDProfessor & Dean of the School of Pharmaceutical SciencesGuangzhou Medical University

2:10 pm Stem Cells and Noncoding RNAs in Cardiovascular DiseasesShijun Hu, PhDProfessor & Deputy Director, Institute for Cardiovascular ScienceFirst Affiliated Hospital, Soochow University

2:20 pm Panel discussion with “Stem Cells & Cardiac Regeneration” speakers

2:40 pm Coffee Break

GENOMICS & POPULATION GENETICSModerator: Joshua Knowles, MD, PhDAssistant Professor of Medicine (Cardiovascular) Stanford University

3:05 pm Genetic Mechanisms of Coronary Artery Disease Thomas Quertermous, MDWilliam G. Irwin ProfessorChief of Cardiovascular Medicine (Research), Stanford

3:15 pm Allele Specific Genome Editing for Inherited DiseasesFeng Lan, PhDProfessor, Deputy Director, Beijing Lab for Cardiovascular Precision MedicineBeijing Anzhen Hospital, Capital Medical University

3:25 pm Cardiac iPSC Biobank to Study Cardiovascular DiseasesJoseph C. Wu, MD, PhD Director, Stanford Cardiovascular InstituteSimon H. Stertzer Professor of Medicine and Radiology, Stanford

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3:35 pm Million Veterans Program, Genomic Sequencing ProjectPhilip S. Tsao, PhDProfessor of Medicine & Associate Chief of Staff (Research)Veterans Administration Palo Alto Health Care System

3:45 pm Opportunities and Challenges for Population and Medical Genomics in the Personal Genome Era

Carlos Bustamante, PhDProfessor and Chair, Department of Biomedical Data Science, Stanford

3:55 pm Panel discussion with “Genomics & Population Genetics” speakers

4:15 pm Closing Comments

4:30 pm Reception, Research Poster Viewing & AwardsJudges: Sean Wu MD PhD, Mark Mercola PhD, Patricia Nguyen MD, Francois Haddad, MD

5:30 pm Adjourn

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1. Right Heart End-Systolic Remodeling Index Strongly Predicts Outcomes in Pulmonary Arterial Hypertension: Comparison With Validated Models

Authors: Myriam Amsallem MD MSa,b, Andrew J. Sweatt MDc, Marie C. Aymami MDa,b, Tatiana Kuznetsova MD PhDd, Mona Selej MDa, HongQuan Lu MDa,b, Olaf Mercier MDe, Elie Fadel MDe, Ingela Schnittger MDa,b, Michael V. McConnell MD MSEEa,b, Marlene Rabinovitch MDf,g, Roham T. Zamanian MDc,f*, Francois Haddad MDa,b,e,f *

Affiliations:a Division of Cardiovascular Medicine, Stanford University, Stanford, CA, USAb Cardiovascular Institute, Stanford University, Stanford, CA, USAc Division of Pulmonary and Critical Care Medicine, Stanford University, Stanford, CA, USAd Research Unit Hypertension and Cardiovascular Epidemiology, KU Leuven Department of Cardiovascular Sciences, Univer-sity of Leuven, Belgiume Division of Cardiothoracic Surgery, Marie Lannelongue Hospital, Le Plessis Robinson, Francef Vera Moulton Wall Center at Stanford, Stanford University, Stanford, CA, USAg Division of Pediatrics, Stanford University, Stanford, CA, USA*Both senior authors contributed equally to this study.

Right ventricular (RV) end-systolic dimensions provide information on both size and function. We investigated whether an internally scaled index of end-systolic dimension is incremental to prognostic scores in pulmonary arterial hypertension. Methods and Results: From 2005 to 2014, 228 patients with pulmonary arterial hyper-tension were prospectively enrolled. RV end-systolic remodeling index (RVESRI) was defined by lateral length divided by septal height. The incremental values of RV free wall longitudinal strain and RVESRI to risk scores were determined. Mean age was 49±14 years, 78% were female, 33% had connective tissue disease, 52% were in New York Heart Association class ≥III, and mean pulmonary vascular resistance was 11.2±6.4 WU. RVESRI and right atrial area were strongly connected to other right heart metrics. Three zones of adaptation (adapted, maladapted, and severely maladapted) were identified based on the RVESRI to RV systolic pressure rela-tionship. During a mean follow-up of 3.9±2.4 years, the primary end point of death, transplant, or admission for heart failure was reached in 88 patients. RVESRI was incremental to risk prediction scores, including the REVEAL score, the Pulmonary Hypertension Connection equation, and the Mayo Clinic model. Using multi-variable analysis, New York Heart Association class III/IV, RVESRI and log NT-proBNP (N-Terminal Pro-B-Type Natriuretic Peptide) were retained (χ2, 62.2; P<0.0001). Changes in RVESRI at 1 year (n=203) were predictive of outcome; patients initiated on prostanoid therapy showed the greatest improvement in RVESRI. Among met-rics, RVESRI demonstrated the best test–retest characteristics. Conclusions: RVESRI is a simple reproducible prognostic marker in patients with pulmonary arterial hypertension.

2. Complementary Value of Heart Failure Risk Scores on Long-term Mortality in Patients Hospitalized with Acute Heart Failure Preserved Ejection Fraction

Authors: Kalyani A Boralkar1, Yukari Kobayashi1, Vedant S Pargaonkar1, Kegan J Moneghetti1, Mirela Tuzovic1, Gomathi Krish-nan1, Sara Bouajila1, Matthew Wheeler1, Dipanjan Banerjee1, Benjamin D. Horne2, Kirk U Knowlton2, Paul A. Heidenreich1, Francois Haddad1

1. Stanford University School of Medicine, Stanford, CA 2. Intermountain Medical Center Heart Institute, Salt Lake City, Utah

Introduction: Heart Failure preserved Ejection Fraction (HFpEF) is a major burden on the healthcare system and better understanding risk prediction in this population may help guide management. Our objective was to assess the complementary value of existing heart failure (HF) risk scores, Get with The Guidelines (GWTG), Intermountain Risk Score (IMRS) and the Meta-Analysis Global Group in Chronic Heart Failure (MAGGIC) Scores

ABSTRACTS

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in an independent cohort of patients hospitalized with acute HFpEF. Methods: Using Stanford Translational Research Integrated Database Environment and individual chart review, we identified 580 adult patients hospitalized with acute heart failure and left ventricular ejection fraction ≥ 50%. We excluded patients with advanced liver/kidney disease, valve replacement, transplantation, active malignancy, pulmonary arterial hypertension and hypertrophic cardiomyopathy. A sub-group of 341 patients had NT-proBNP levels available. Mortality status was determined using United States Social Security Death Index and chart review. Cox pro-portional hazard analysis was used to determine the HF risk scores predictive of all-cause mortality in HFpEF. Results: The mean age was 76 ± 15 years with 334 (57.6%) females. Median length of hospital stay was 4 (IQR 2 – 9) days and follow up of 2.0 (IQR 0.2 – 4.6) years with a total of 140 (24.1%) deaths. On multivariate analysis, the GWTG, IMRS and MAGGIC scores were independent correlates of all-cause mortality with normalized haz-ard ratios of 1.48 (1.17 – 1.86), 1.41 (1.15 – 1.76) and 1.31 (1.04 – 1.70) respectively. In patients with NT-proBNP collected, NT-proBNP remained significantly associated with mortality with normalized hazard ratio of 1.52 (1.22 – 1.91) even after adjustment for the risk scores. Conclusion: In patients hospitalized with acute HFpEF, established heart failure Risk scores together with NT-proBNP can play a complementary role in outcome prediction.

3. Telomere Shortening as a Hallmark of Lethal Dilated Cardiomyopathy

Alex C Chang1,4, John W Day2, Joseph C Wu3,4, Helen M Blau1,4

1. Baxter Laboratory for Stem Cell Biology, Microbiology and Immunology, Stanford, CA,2. Department of Neurology, Medicine, Stanford, CA3. Division of Cardiology, Medicine, Stanford, CA4. Stanford Cardiovascular Institute, Medicine, Stanford, CA

Introduction: Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disease that results from mutations in the dystrophin gene and is the most common myopathic disease in humans. Dystrophin is crucial for the formation of a dystrophin-glycoprotein complex, which connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix in both skeletal and cardiac muscles. In the heart, a loss of dystrophin leads to increased fibrosis and death in the third decade of life due to dilated cardiomyopathy. A conundrum in studying and developing therapies for DMD has been the lack of a mouse model that fully recapitulates the clinical phenotype. When we ‘humanized’ the mdx mouse by creating a novel mouse model with shortened telomere lengths, both the skeletal muscle and cardiac muscle phenotypes of DMD culminating in lethal dilated cardiomyopathy were fully recapitulated. Moreover, we found that the cardiomyocytes in the hearts of four patients with DMD had telomeres with 50% the length of unaffected controls. Notably, telomere lengths in smooth muscle cells within the same cardiac tissues are unaffected, serving as robust internal controls for the specificity of telomere shortening to those cells requiring dystrophin for function. Hypothesis: We hypothe-size that contractile defects due to dystrophin deficiency drive telomere shortening and result in metabolic compromise and cell death due to inhibition of mitochondrial function and biogenesis. Method: To study telomere shortening in disease progression, we generated three DMD human induced pluripotent stem cell lines and differentiated them into beating cardiomyocytes (hiPSC-CMs). Results: Our data reveal progressive telomere shortening in non-dividing hiPSC-CMs that leads to a marked DNA damage response culminating in mitochondrial dysfunction. Conclusions: Patient hiPSC-CMs recapitulate in 30 days the telomere shortening that occurs in 30 years of life in DMD patients and this technology enables the study of cause and effect and tests of interventions.

4. Diagnostic Performance of Resting Distal to Aortic Coronary Pressure Using Instantaneous Wave-Free Ratio as a Reference Standard

Yuhei Kobayashi, Nils P. Johnson, Frederik M. Zimmermann, Nils Witt, Colin Berry, Allen Jeremias, Bon-Kwon Koo, Giovanni Esposito, Gilles Rioufol, Seung-Jung Park, Takeshi Nishi, Dong-Hyun Choi, Keith G. Oldroyd, Emanuele Barbato, Nico H.J. Pijls, Bernard De Bruyne, William F. Fearon, MD

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Background: Recently, two randomized controlled trials suggested that the instantaneous wave-free ratio (iFR), a resting coronary physiologic index is non-inferior to fractional flow reserve for guiding revasculariza-tion. The distal to aortic coronary pressure (Pd/Pa) measured at rest is another adenosine-free index widely available in the cardiac catheterization laboratory; however, little is known about the diagnostic performance of Pd/Pa using iFR as a reference standard. Methods: A total of 763 patients were prospectively enrolled from 12 institutions. iFR and Pd/Pa were measured under resting conditions. Using iFR≤0.89 as a reference stan-dard, the diagnostic performance of Pd/Pa and its best cutoff value were assessed. Results: By independent core laboratory analysis, iFR and Pd/Pa were analyzable in 627 and 733 patients (82.2% vs. 96.1%, p<0.001), respectively. The median iFR and Pd/Pa were 0.90 (interquartile range 0.85-0.94) and 0.92 (0.88-0.95) and the two indices were highly correlated (R2 = 0.93, p<0.001, iFR = 1.31*Pd/Pa-0.31). By receiver operating charac-teristics curve analysis, Pd/Pa showed excellent diagnostic performance (area under the curve = 0.98, 95% confidence interval 0.97 to 0.99, p<0.001) with a best cutoff value of Pd/Pa≤0.91. The diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value were 93.0%, 91.4%, 94.4%, 93.3%, and 92.7%, respectively. These results were similar in patients with acute coronary syndrome and stable angina. Conclusions: Pd/Pa was analyzable in a significantly higher number of patients than iFR. Pd/Pa showed excellent diagnostic performance against iFR and would likely result in similar outcomes as iFR, if used to guide revascularization.

5. Long-Term Prognostic Value of Invasive and Non-Invasive Measures Early after Heart Transplantation

Yukari Kobayashi, MD; Yuhei Kobayashi, MD; Hyoung-Mo Yang, MD; Helen Luikart, RN; Takeshi Nishi, MD; Dong-Hyun Choi, MD; Ingela Schnittger, MD; Hannah A. Valantine, MD; Kiran K. Kush, MD; Alan C.Y. Yeung, MD; Francois Haddad, MD; William F. Fearon MDStanford Cardiovascular Institute and Stanford Cardiovascular Medicine

Background: Invasively assessed coronary microvascular resistance early after heart transplantation (HT) pre-dicts worse long-term outcome; however, little is known about the relationship between microvascular resis-tance, left ventricular function and outcomes in this setting. Methods: A total of 100 cardiac transplant recipients had fractional flow reserve (FFR) and the index of microcirculatory resistance (IMR) measured in the left anterior descending artery and echocardiographic assessment including left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS) at 1 year after HT. The primary endpoint was the cumulative survival free of death or retransplantation after 1 year. Results: The mean FFR, IMR, LVEF, and GLS values at 1 year were 0.87±0.06, 21.3±17.3, 60.4±5.4%, and 14.2±2.4%, respectively. FFR had no significant correlation with LVEF (p = 0.94) and GLS (p = 0.86). Similarly, IMR had no significant correlation with LVEF (p = 0.54) and GLS (p = 0.90). During a mean follow-up of 6.7±4.2 years (7.7 years after transplant), the primary endpoint occurred in 24 patients (24.0%). By ROC curve analysis, IMR = 19.3 and GLS = 13.3% were the best cutoff values for predicting death or retransplanta-tion. The cumulative event-free survival was significantly lower in patients with higher IMR (log-rank p = 0.02) and lower GLS (log-rank p<0.001). The cumulative event-free survival could be further stratified with the combina-tion of IMR and GLS (p<0.001). By multivariate Cox-proportional hazards model, higher IMR and lower GLS were independently associated with the long-term death or re-transplantation (elevated IMR, HR = 2.50, p = 0.04 and reduced GLS, HR = 3.79, p = 0.003). Conclusions: Invasively assessed coronary microvascular resistance (IMR) does not correlate with left ventricular systolic function (GLS) at 1 year after heart transplantation. IMR and GLS determined at 1 year are independent predictors of late death or retransplantation.

6. Single Cell Profiling Identifies INO80 as a Critical Regulator Dictating Cardiac Lineage Commitment

Yingnan Liao1, Peng Yu, PhD1, Zongna Ren1 , Li Wang, MD, PhD1

1 State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China.Correspondence: wangl@pumc.edu.cn

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During human heart development, multiple molecular signaling pathways drive fate decisions. Here, we present a study of early human heart development using single-cell transcriptomics and a 2D in vitro heart development model. We computationally identify INO80 complex accompanied cardiogenesis and experimen-tally confirm the predictions. Knockdown of Ino80 represses genes that are implicated in cardiac differentia-tion. Ino80 localizes to cardiac specific regulatory regions, impacting cardiac differentiation gene expression. In conclusion, INO80 may alter the cardiac epigenome, instructing gene expression changes that drive cardiac differentiation.

7. Spatially Patterned Atheroprotective Vascular Grafts for Enhanced Patency

Karina H. Nakayama1,2, Masashi Kawamura2,3, Hanway Wang1, Joseph Woo2,3, Ngan F. Huang1,2,3

1 Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, CA, USA2 The Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA3 Department of Cardiothoracic Surgery, Stanford University, Stanford, CA, USA

With 25 million American suffering from at least one clinical manifestation of atherosclerosis, and approx-imately 500,000 deaths annually attributed to coronary artery disease, there is a pressing need to better understand the biological basis for the development of atherosclerosis. Our previous work demonstrated that spatial patterning cues from extracellular matrices (ECMs) play an important role in modulating endo-thelial cell (EC) angiogenic phenotype including inhibiting adhesion of lipogenic proteins and monocytes in the circulating blood that leads to atherogenesis. Therefore, we hypothesized that vascular grafts containing longitudinally oriented spatial patterning will promote aligned re-endothelialization to provide atheroprotec-tive function and improved graft patency, when compared to vascular grafts without cell patterning. Ran-domly-oriented or aligned vascular grafts (0.5 cm length, 1 mm diameter) were fabricated from electrospun poly (ε-caprolactone) using a dual layer approach. Heparin was conjugated to the grafts and demonstrated a release rate of 202 μg per scaffold over a 48h period. Transplantations of heparinized acellular grafts into the rat carotid artery demonstrated patency by B-mode echo 24h post transplant. Studies are currently underway to assess long term patency and histology of grafts transplanted into the rat carotid artery. Findings from these studies will provide novel insights into the mechanisms of resisting atherogenesis that are mediated by spatial cell patterning, and have important translational potential in the generation of atheroprotective vascular grafts that enhance graft patency.

8. Clinical Outcome and Long-term Follow-up in Patients with Angina in the Absence of Obstructive CoronaryArtery Disease

Authors: Vedant S Pargaonkar MD1, Yuhei Kobayashi MD1, Takumi Kimura MD PhD1, Ingela Schnittger MD1, Ian S Rogers MD MPH1, David P Lee MD1, William F Fearon MD1, Alan C Yeung MD1, Marcia L Stefanick PhD2, Jennifer A Tremmel MD MS1

1. Stanford Cardiovascular Institute, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford2. Department of Medicine, Stanford University School of Medicine, Stanford CA

Introduction: A comprehensive invasive evaluation of patients with angina in the absence of obstructive coro-nary artery disease (CAD) may identify occult coronary abnormalities contributing to symptoms, and may also help guide therapy. However, whether this has an impact on symptoms and quality of life (QOL) is unclear. Our objective was to study the long-term follow-up of anginal symptoms in patients with endothelial dysfunction, microvascular dysfunction (MVD), and/or a myocardial bridge (MB). Methods: Before and after invasive testing, we administered a Seattle angina questionnaire (SAQ) to 157 women and 48 men found to have no obstructive CAD on angiography. Endothelial dysfunction was defined as a decrease in luminal diameter of >20% after intracoronary (IC) acetylcholine, MVD was defined as an index of microvascular resistance ≥ 25, and an MB was defined as an echolucent half-moon sign and/or ≥10% systolic compression on intravascular ultrasound.Results: Median follow up was 1.9 (1.7 – 2.2) years. Endothelial dysfunction was present in 113 (55%) patients,

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MVD in 47 (23%), and MB in 93 (45%). Medical treatment and lifestyle changes were suggested according to the diagnosis of the testing. Eleven had surgical unroofing of an MB. There were no MACE including MI, revas-cularization, or stroke. There were 2 deaths (both cancer). There was a significant improvement in physical limitations due to angina, anginal stability, anginal frequency, treatment satisfaction, and QOL (p < 0.001). On multivariable logistic regression analysis, arterial compression after IC acetylcholine, post-invasive testing cal-cium channel blocker usage, and age were independent predictors of significant improvement in SAQ scores. Conclusion: A comprehensive invasive evaluation can identify occult coronary abnormalities in patients with angina in the absence of obstructive CAD and help guide therapy, which ultimately results in an improvement in patients’ symptoms and QOL.

9. RNASeqFPro, a Pipeline for RNA-Seq Differential Gene Expression Analysis Using Transcription Noise Model

Milos Pjanic1,2, Clint L. Miller1,2, Thomas Quertermous1,2

1. Division of Cardiovascular Medicine, Stanford University, Stanford, California, United States of America.2. Cardiovascular Institute, Stanford University, Stanford, California, United States of America.

RNA-Seq analysis comprises multiple steps, including quality control, mapping, read counting and differential expression (DE) analysis. Here we present a pipeline for automated RNA-Seq DE analysis based on top isoform selection. RNASeqFPro can perform analysis on paired-end or single-read RNA-Seq data for mouse mm10 and human hg19 genomes. RNASeqFPro sorts and pairs fastq files, then, performs FastQC quality control, and maps fastq files to the reference genomes using STAR second pass mapping. It uses featureCounts to perform read summarization on the GENCODE mouse or human transcript collection. It calls fileMulti2TableMod1, an awk script we developed to generate the mastertable and writes an R script to select the top transcript and converts GENCODE to RefSeq identifiers using biomaRt. Finally, it writes an R script and performs DESeq analy-sis of differential gene expression, creates output tables and generates graph outputs using GO term annota-tions and goseq package. We also created two versions that use Kallisto for fast pseudo-alignment of the reads to the GENCODE transcripts that dramatically reduced processing time. Both STAR and Kallisto versions of RNASeqFPro are based on the transcription noise model and selection of the top expressed transcript for dif-ferential gene expression (DGE) analysis, therefore considering other transcripts rising from the same locus as transcriptional noise not contributing to the effective gene expression level. RNASeqFPro performs all steps in a sequential manner, and thus significantly saves preparation time for each run. RNASeqFPro should be useful for researchers with less computational experience as it requires a single executing command.

10. Rapid remodeling of an autologous acellular conduit into an integrated neoartery

Xuefeng Qiu1, 2, 3, Benjamin Li-Ping Lee2, Sze Yue Wong2, Ryan Sochol4, Nianguo Dong1, Song Li2, 3, 5

1 Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Tech-nology, Wuhan, Hubei 430022, China2 Department of Bioengineering, University of California, Berkeley, CA 94720, USA3 Department of Bioengineering, University of California, Los Angeles (UCLA), Los Angeles, CA90095, USA4 Department of Mechanical Engineering, University of Maryland, College Park, MD 20742, USA5 Department of Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA

Vascular grafts formed by decellularized matrix have shown promising functional results in early clinical trials. However, graft failures caused by poor remodeling and pro-inflammatory immune responses are still unavoidable, and the remodeling mechanisms are also unclear. Here we developed a novel decellularized autologous vascular graft (DAVG) with looser fibers, which further was implanted as an interpositional graft in the common carotid artery. The key advantages of this methodology include: constructive immune response leading to extremely high patency rates, uniform remodeling with complete infiltration of cells into the whole graft to generate a neoartery exhibiting superiorly similar structure to a native artery. Autologous stem/pro-genitor cells were involved in the graft remodeling. Additional preliminary in vivo studies in the minipig model

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returned encouraging results. Taken together, this approach can be quickly translated to patients and will have an impact on the regeneration of autologous tube type organs in the future. Funding: This work is supported in part by the grants from National Natural Science Foundation of China (81170110/H0203 to X.Q.), National Key R&D Plan (2016YFA0101100 to N.D.), the National Institute of Health (EB012240 and HL083900 to S.L.) and California Institute for Regenerative Medicine (A clinical fellow’s training grant TG2-01164 to X.Q.).

11. Ino80 Regulates Cardiac Hypertrophy and Remodeling in Response to Stress

Zongna Ren, MS1, Peng Yu, PhD1, Yingnan Liao1 , Li Wang, MD, PhD1

1 State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China.Correspondence: wangl@pumc.edu.cn

Cardiac hypertrophy, a complex and dynamic process involving multiple factors, leads to heart failure ultimately, but the mechanism of its development has not yet been fully elucidated. The INO80 chromatin remodeling enzyme has roles in transcription, DNA repair and replication. INO80 facilitates pluripotency gene activation in embryonic stem cell self-renewal, reprogramming, and blastocyst development. Interestingly, INO80 has also been identified as a pivotal factor in maintaining normal heart function in zebra fish. Howev-er, the role of INO80 in cardiac hypertrophy remains unclear. This project will clarify the role and mechanism INO80 in cardiac hypertrophy at cellular, animal and molecular levels. First, we will generate LentiIno80 and LentishIno80 lentivirus to infect neonatal rat cardiomyocytes which are induced by PE to investigate the role and mechanism of Ino80 in cardiac hypertrophy at the cellular level. Secondly, we will generate a Ino80 knock-out mouse model and a TG mouse model with cardiac-specific Ino80 expression which would be subjected to aortic banding surgery to clarify the role and mechanism of INO80 in cardiac hypertrophy. In the third part, we will block or activate the key downstream protein of INO80 via drugs or genetic engineering to reverse the phenotype of Ino80 knockout or transgenic mice. We aim to clarify the role and mechanism of INO80 in cardiac hypertrophy to provide new molecular targets for the prevention and therapy cardiac hypertrophy.

12. Endothelial Deletion of Ino80 Disrupts Coronary Angiogenesis and Causes Congenital Heart DiseasePhenotypes

Siyeon Rhee1, 2, Jae I. Chung1, 2, Devin A. King1, Anna Duan1, Gaetano D’amato1, Andrew Chang1, Bikram Sharma1, Youngtae Jeong3, 4, 5, Maximilian Diehn3, 4, 5, Ashby J. Morrison1* & Kristy Red-Horse1*

1 Department of Biology, Stanford University, 371 Serra Mall, Stanford, California 94305, USA.3 Stanford Cancer Institute, Stanford University School of Medicine, Stanford, California, 94305, USA. 4 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, 94305, USA. 5 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, 94305, USA.

Organ size control is an important process in developmental biology that remains incompletely understood. This applies to the mammalian heart, where the determinants that support ventricle wall expansion are not fully characterized. Further knowledge could provide insight into devastating human diseases such as Left Ventricular Non-Compaction (LVNC). We found that deleting the Ino80 chromatin remodeler from endothelial cells resulted in an LVNC-like phenotype (thin compact myocardium), which correlated with defective coronary vascularization. Deletion of Ino80 individually in the two major coronary progenitors resulted in intermediate non-compaction phenotypes. In vitro, endothelial cells promoted ventricular muscle expansion independent of oxygen delivery in an Ino80-dependent manner. Mechanistically, loss of Ino80 resulted in increased E2F-me-diated gene expression and S-phase progression. These data support a model where Ino80-mediated control of cell cycle genes is required for coronary angiogenesis. Furthermore, providing two blood vessels sources accelerates myocardial expansion, detexrmining heart wall size and suppressing disease phenotypes.

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13. Modulation of Cardiomyocyte Physiology by Stretch-induced miRNAs

Christine Wahlquist1, Agustin Rojas-Muñoz1, Fabio Cerignoli2, Ricardo Serrano2, Wienand Omta3, Ranjan Perera4, David Egan3, Juan Carlos del Alamo2 & Mark Mercola1

1 Stanford University, Cardiovascular Institute, Palo Alto, CA. 2 UCSD, Department of Bioengineering, La Jolla, CA. 3Universi-ty Medical Center Utrecht, Department of Cell Biology, Utrecht. 4Sanford Burnham Prebys, Orlando, FL.

The pathological decline in cardiac function observed during heart failure (HF) correlates with the inability to match contraction with workload at the cellular level. Cardiomyocyte contractility is the result of a coordinat-ed integration of sarcolemma depolarization, Ca2+ handling, energy production and the structural contractil-ity network. Acute pathological stimuli can induce alterations in these processes, making the cardiomyocyte susceptible to contractile dysfunction and arrhythmias, often culminating in HF. However, the mechanisms connecting acute stimuli to the early maladaptive signaling that dictates disease progression remain poor-ly understood. miRNAs are frequently dysregulated in HF, suggesting a potential role in early remodeling processes. To explore this possibility, we have focused on microRNA control of contractility in the context of hemodynamic stress. We developed a comprehensive approach to deconstruct the interactions between the miRNA landscape and their potential targets during cyclic mechanical stretch. This pipeline has revealed a unique miRNA signature associated with acute stretch, confirmed their individual ability to affect contractile velocity, sarcomere integrity and metabolic function in cardiomyocytes, and identified direct biophysical mRNA targets through AgoIP RISC-Seq analysis. Our approach focusing on acute mechanical stimuli explores previously overlooked target space that may be of therapeutic relevance for early intervention strategies.

14. Imaging Transparent Intact Cardiac Tissue with Single-Cell ResolutionZhiwei Wang, PhD candidate1; Jie Zhang, PhD1; Guangpu Fan, MD, PhD2; Hui Zhao, PhD1; Xu Wang, MD1; Jing Zhang, MD1; Peide Zhang, MD1; Wei Wang, MD, PhD1

1 Cardiovascular Surgery Department, State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; 2Department of Cardiovascular Surgery, Peking University People’s Hospital, Beijing, China.Correspondence: drweiwang0728@hotmail.com (Wei Wang)

Introduction: Tissue clearing methods, together with immunofluorescence and modern imaging techniques, have broken the limits of traditional research, upgrading histological study from a 2D to a 3D scale. Due to the distinct structures of different organs, the clearing conditions differ. Most of the clearing agents were designed for brain tissues, the specific clearing conditions and agents for cardiac tissues have never been studied up until now, and 3D microstructures of intact heart tissues as well as fibroblasts phenotype changes after myocardial infarction remodeling have not been displayed at an integral level. Methods & Materials: We used mouse cardiac sections to look for a specific cardiac tissue clearing condition, and circulation perfusion was tested for whole-heart clearing. Immunofluorescence and 3D reconstruction were used to locate the relative space distribution of different molecules in cardiac tissues and to show dynamically the fibroblasts phenotype changing after myocardial infarction in a stereoscopic pattern. For immunofluorescence, the role of electric force was explored to achieve fast antibody labeling. Conclusions: A new convenient and effective cardiac tissue clearing agent SUT (Scheme Update on tissue Transparency) was developed, and whole-heart clearing of mouse, rat and pig were achieved by circulation perfusion. The relative 3D space distribution of different molecules were shown, while the procession of fibroblast phenotype changes were displayed at an integral level with single-cell resolution. EAL (Electrophoretic Antibody Labeling) was created for fast antibody labeling to acquire high-efficiency immunofluorescence.

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15. The Contraction and Maturity of Cardiomyocytes Derived from Pluripotent Stem Cells is Influenced byScaffold Microtopography

Maureen Wanjare1,2, Luqia Hou1,2, Karina H. Nakayama1,2, Joseph J. Kim1,2, Nicholas P. Mezak1, Oscar Abilez2, Evangeline Tza-tzalos2, Joseph C. Wu2, and Ngan F. Huang1,2,3

1 Center for Tissue Regeneration, Repair and Restoration, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA. 2 Stanford Cardiovascular Institute, Stanford University, Stanford, CA, USA 3 Department of Cardiothoracic Surgery, Stanford University, Stanford, CA, USA

Over 15 million people suffer from coronary heart disease (CHD), which is characterized by narrowing of the coronary arteries that supply blood flow to the heart, which may ultimately lead to heart failure. Current cell-based clinical trials to restore cardiomyocyte (CM) health by local delivery of cells have shown only moderate benefit in improving cardiac pumping capacity. Engineering of myocardial tissue constructs is a promising approach for treatment of coronary heart disease. To engineer myocardial tissues that better mimic the highly ordered physiological arrangement and function of native cardiomyocytes, we generated electrospun micro-fibrous polycaprolactone scaffolds with either randomly oriented or parallel-aligned microfiber arrangement. Additionally, the scaffolds were co-seeded with human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and endothelial cells (iECs) for up to 12 days after iCM seeding. We demonstrated that aligned microfi-brous scaffolds induced iCM alignment along the direction of the aligned microfibers and promoted greater iCM maturation by increasing the sarcomeric length and gene expression of myosin heavy chain adult isoform (MYH7), in comparison to randomly oriented scaffolds. Anisotropy also promoted significantly higher maxi-mum contraction velocity of iCMs cultured after 12 days, compared to randomly oriented scaffolds. Co-seeding of iCMs with iECs on scaffolds led to reduced contractility, compared to when iCMs were seeded alone. These findings demonstrate a dominant role of scaffold anisotropy in engineering cardiovascular tissues that main-tain iCM organization and contractile function.

16. Studying Cardiovascular Effects of Marijuana on Healthy Individuals Using Human-derived InducedPluripotent Stem Cells

Tzu-Tang Wei1,2,3, Ian Y. Chen1,2,3, Vincent Ho1,2,3, Jayakumar Rajadas4, Joseph C. Wu1,2,3

1 Stanford Cardiovascular Institute2 Department of Medicine and Radiology3 Institute of Stem Cell Biology and Regenerative Medicine4 Biomaterials and Advanced Drug Delivery (BioADD) Laboratory of Pharmacology Division, Stanford University School of Medicine, Stanford, CA 94305, USA.

Marijuana is the most widely used illicit drug worldwide. In the US, eight states have legalized recreational marijuana use to date, including California. The recreational use of marijuana is also expanding rapidly in Asia and Europe. However, compared to other widely used drugs such as alcohol or cigarettes, less is known about its adverse effects. Epidemiological studies indicate that marijuana smoking increased the risk of coronary artery disease (CAD). Adverse cardiovascular, cerebrovascular, and peripheral vascular effects also have been reported to be associated with marijuana use. These studies suggest that marijuana use may impair vascular endothelial function. Here we investigate the effect of Δ9-tetrahydrocannabinol (Δ9-THC) on vasculature using human induced pluripotent stem cell-endothelial cells (iPSC-ECs). Δ9-THC induced cytotoxicity in iPSC-ECs and human endothelial cells, without showing apparent cytotoxicity towards cardiac fibroblasts and cardio-myocytes. The mRNA expression of inflammation, oxidative stress, angiogenesis, and migration-related genes were induced in iPSC-ECs after Δ9-THC treatment. Δ9-THC-induced inflammation persisted 8-10 days. These results suggest that Δ9-THC causes endothelial activation in iPSC-ECs. In addition, endothelial dysfunction was found in Δ9-THC-treated iPSC-ECs, with induced inflammation, increased oxidative stress, and impaired angiogenesis and migration abilities. We found genistein, a flavonoid abundantly present in soybeans, could

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attenuate Δ9-THC-induced endothelial activation and dysfunction in iPSC-ECs. Taken together, these results indicate that marijuana use cause endothelial activation and dysfunction in healthy individuals, thereby increasing the risk for CAD and vascular diseases. Genistein shows potential in the prevention of marijuana-im-paired vascular endothelial function.

17. Restore Impaired Diastolic Function in Induced Pluripotent Stem Cell Derived Cardiomyocytes From Hypertrophic Cardiomyopathy Patients

Haodi Wua,b,c, Huaxiao Yanga,b,c Joe Zhanga,b,c, Chi Keung Lama,b,c, June-Wha Rheea,b,c, Alex CY Changad, Timon Seegera,b,c, Karim Sallama,b,c, Ning Maa,b,c, Donald M. Berse, Joseph C. Wua,b,c

a) Stanford Cardiovascular Institute; b) Department of Medicine, Division of Cardiology; c) Institute for Stem Cell Biology and Regenerative Medicine; Stanford University School of Medicine, Stanford, CA 94305, USA; d) Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology; e) Department of Pharmacology, University of California, Davis, CA 95616, USA

Diastolic dysfunction (DD) is commonly seen in hypertrophic cardiomyopathy (HCM), yet the cellular mech-anism of DD is not fully understood, and no specific treatment so far has been developed. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold the potential for disease modeling, mechanism study and drug screening of DD in HCM. In the current study, beating iPSC-CMs were generated from healthy and DD patients carrying familial HCM mutations (MYH7 R663H, TPM1 N835H, and MYBPC3 V321M). Sarcomere shortening measurement with micropatterned iPSC-CMs showed impaired diastolic function as prolonged relaxation time and reduced relaxation rate in HCM cells. Fura-2 AM calcium imaging in-dicated elevated diastolic calcium and abnormal calcium handlings in HCM iPSC-CMs, which were exaggerated by the β-adrenergic challenge. Combining calcium handling and traction force microscopy (TFM), we found the index of calcium sensitivity (measured as dF/Δ[Ca2+]i) was enhanced by HCM mutations. Thus, both diastolic calcium overload and myofilament sensitization lead to impaired relaxation of HCM cells. To retain diastolic function in HCM iPSC-CMs, we restored calcium homeostasis in diseased cells by partially blockade of the cal-cium or late sodium current. Our results show the treatments have reset the diastolic calcium level, restored the diastolic function, restricted CaMKII activity and improved long-term survival of HCM iPSC-CMs, suggesting calcium signaling remodeling as an important intracellular pathological mechanism of DD. In summary, the current study developed HCM patient specific iPSC-CM models that recapitulated DD at single cell level, and revealed novel cellular mechanisms and potential therapeutic targets of DD in HCM cardiomyocytes.

18. Topical Application of IcyHot Reduces Myocardial Infarct Size in Rodents by a TRPA1-dependent Mechanism

Yun Wu, MD, Yao Lu, MD, PhD, Eric R. Gross, MD, PhDDepartment of Anesthesiology, Perioperative and Pain Medicine, Stanford University, School of Medicine, Stanford, CA; Correspondence: ergross@stanford.edu

Background: Toxic reactive aldehydes are formed during ischemia-reperfusion. The ion channel transient receptor potential ankryin1 (TRPA1) is irreversibly modified by reactive aldehydes which can cause calcium influx and cell death. Here we tested whether topically applied creams containing a reversible TRPA1 agonist could reduce ishchemia-reperfusion injury. Methods: Male Sprague-Dawley rats 8-10 weeks age were subjected to an in vivo myocardial ischemia-reperfusion model of 30 minutes of left anterior descending (LAD) coronary artery ischemia followed by 2 hours reperfusion. Prior to ischemia, rats were untreated or had 1g of cream applied to the abdomen. The creams tested were IcyHot, Bengay, Tiger Balm, or Preparation H. The heart were negatively stained for the area at risk and the infarct size was determined by using TTC staining. A subset of rodents also received an intravenous bolus of the TRPA1 antagonist TCS-5861528 (1mg/kg) or AP-18 (1mg/kg) prior to receiving IcyHot. Further, we isolated adult rat cardiac myocytes and subjected the cardiac myocytes to hypoxia-reoxygenation to evaluate the effect of methyl salicylate (an active ingredient of IcyHot).Results: Interestingly, both IcyHot and Bengay reduced myocardial infarct size compared to untreated rodents

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(IcyHot: 41±3%*, Bengay: 50±2%*, vs control: 62±1%, infarct size/area at risk%, n=6/group, *P<0.05). Giving a TRPA1 antagonist prior to IcyHot blocked the reduction in infarct size. In isolated cardiac myocytes, methyl salicylate (MS), an ingredient of IcyHot, reduced cardiac myocyte cell death by percentage of lactate dehydro-genase (LDH) release when given during reoxygenation (MS 1mM: 33±5%*, MS 2mM: 26±7%* vs DMSO: 59±9%, n=9/group, *P<0.05). The plasma concentration of salicylate, which is metabolite of MS in IcyHot increased during the reperfusion. Conclusion: Since IcyHot and Bengay are safely used by humans, products such as these could be quickly translatable and widely used to reduce ischemia-reperfusion injury.

19. The Role of Collagen Nanofibrillar Alignment in Endothelial Cell Function and Survival

Guang Yang1,2, Maureen Wanjare1, 2, Karina Nakayama1, 2, Tatiana Zaitseva4, Michael V. Paukshto4, Ngan F. Huang1,2,3 1Stanford University, Department of Cardiothoracic Surgery, 2Stanford Cardiovascular Institute, 3VA Palo Alto Health Care System, Palo Alto, CA, 4Fibralign Corporation, Union City, CA

Research Background & Objectives: Endothelial cells (ECs) are a promising cell type for the treatment of peripheral arterial disease (PAD). Our lab previously showed that anisotropic nanofibrillar scaffolds augment the angiogenic function of human ECs. In order to optimize the biomechanical properties of nanofibrillar scaf-folds, we examined the effect of nanofibril size and crosslinking on endothelial survival and function.Methodology: Aligned nanofibrillar collagen scaffolds were fabricated using a shear-based method. Scaffold formulations of varying fibril diameters (90-160 nm) were prepared by altering the ionic strength of monomer-ic collagen. Scaffolds were further crosslinked at varying levels, and characterized for surface topography and degradation rate. Human ECs were seeded onto the scaffolds for assessment of migration and survival under hypoxia. Results: Scaffold formulation with low ionic strength presents low diameter (LD) fibrils (~90 nm), com-pared to the high ionic strength formulation group (~160 nm). HD scaffold possesses higher elastic modulus than LD group (20.74 ± 11.73 mPa vs. 13.23 ± 6.17 mPa). The mean direction of migration with respect to the fi-bril axis was 37.06° on HD, compared to 48.45° on LD, regardless of the degree of crosslinking. Cell survival was ~11% higher on HD scaffolds than that on LD group after culture in 1% O2 for 3 days to mimic tissue ischemia. These data suggest that aligned HD scaffolds promote EC survival and alignment along the fibril direction.Significance: This study provides new insight into the role of biophysical microenvironment in the survival, migration and angiogenesis of ECs. The findings may lead to a novel clinical strategy for the therapeutic neo-vascularization of PAD patients.

20. Novel In Vivo miRNA Delivery with Nanoparticles by Injectable Hydrogel for Cardiovascular Function Improvement

Huaxiao Yang,1 Kai Li,2, 3 Huiyuan Wang,4 Xulei Qin,1 Xin Zhao,1 Haodong Chen,1 Yonggang Liu,1 Hung-Ta Wo,1 Katherine Chung,1 Sarah C. Heilshorn,4 and Joseph C. Wu.1, 5,*1Stanford Cardiovascular Institute, Stanford, CA, 94305, United States; 2Department of Radiology, Stanford, CA, 94305, Unit-ed States; 3Institute of Materials Science and Engineering, A*STAR, Singapore, 117602, Singapore; 4Department of Materials Science and Engineering, Stanford, CA, 94305, United States; 5 Department of Medicine, Division of Cardiology, Stanford, CA, 94305, United States. *Address correspondence to joewu@stanford.edu.

Cardiovascular disease (CVD) is the No. 1 killer in the United States, and one of leading causes is heart failure caused by myocardial infarction (MI). A number of new approaches have been reported to restore the wors-ening condition of infarcted myocardium via either exogenous stem cell transplantation, or growth factor and miRNA based interventions to stimulate the intrinsic rejuvenating mechanisms. In this study, we have estab-lished a novel in vivo delivery system with nanoparticles (NPs) as miRNA carriers and elastin-based injectable hydrogel as vehicle to transport the miRNA into the infarcted myocardium to achieve cardiovascular function improvement. First, the NPs with core-shell structure at ~100 nm in diameter has been successfully synthe-sized to encapsulate the miRNA with intact length and function. Then, the uptake efficiency of NP in human

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embryonic stem cell -cardiomyocyte (hESC-CMs) and -endothelial cells (hESC-ECs) were higher than 60% at varied loading concentrations showing less toxicity than that of the lipofectamine RNAiMAX transfection. The in vivo rat study with NP/hydrogel injections demonstrated the improved cardiac function with increased ejection fraction (EF, echo) by 20%, smaller scar size by 10%, and higher capillary density in the scar area in 4 weeks. Further, the hESC-CM and hESC-EC in vitro modeling were utilized to confirm the cellular and molecu-lar mechanisms on the cardiovascular function improvement. In summary, the novelty of this in vivo miRNA delivery system via NPs and injectable hydrogel stands on the high transfection efficiency and cell viability of cardiovascular cells and fast cardiac contraction adaption of the hydrogel in a very convenient therapeutic approach.

21. Myocardial Reparative Functions of Exosomes From MSCs are Enhanced by Hypoxia Treatment of the Cells via Fransferring microRNA-210

Jinyun Zhu, Kai Lu, Jian’an Wang, Hong YuDepartment of Cardiology, Second Affiliated Hospital, College of Medicine, Zhejiang University. 88 Jiefang Rd, Hangzhou, Zhejiang Province, 310009, PR China

Hypoxia treatment enhances paracrine effect of mesenchymal stem cells (MSCs). The aim of this study was to investigate whether exosomes from hypoxia-treated MSCs (ExoH) are superior to those from normoxia-treated MSCs (ExoN) for myocardial repair. Mouse bone marrow-derived MSCs were cultured under hypoxia or normox-ia for 24 hours, and exosomes from conditioned media were intramyocardially injected into the infarcted heart of C57BL/6 mice. ExoH resulted in significantly higher survival, smaller scar size and better cardiac functions recovery. ExoH conferred increased vascular density, lower cardiomyocyte apoptosis, reduced fibrosis, and increased recruitment of cardiac progenitor cells in the infarcted heart relative to ExoN. MicroRNA analysis revealed significantly higher levels of microRNA-210 (miR-210) in ExoH compared with ExoN. Transfection of a miR-210 mimic into ECs and CMs conferred similar biological effects as ExoH. Hypoxia treatment of MSCs increased the expression of neutral sphingomyelinase 2 (nSMase2) which is crucial for exosome secretion. Blocking the activity of nSMase2 resulted in reduced miR-210 secretion and abrogated the beneficial effects of ExoH. In conclusion, hypoxic culture augments miR-210 and nSMase2 activities in MSCs and their secreted exo-somes, and this is responsible at least in part for the enhanced cardioprotective actions of exosomes derived from hypoxia-treated cells.

22. Modified mRNA Transcript-incorporated Alginate Scaffold as Controlled Delivery System for Angiogenic Factors mRNA, for Revascularization of Ischemic Skeletal Muscles

Maedeh Zamani1, Ngan F. Huang1,2

1 Department of Cardiothoracic Surgery, Stanford University, Stanford, CA, USA; 2 Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA

Massive muscle injuries caused by traumatic or surgical actions may significantly impair the regeneration and revascularization capability of skeletal muscle. As an alternative to reconstructive surgery, biomimetic scaf-folds capable of providing an efficient platform for endogenous muscle regeneration and potentially inducing revascularization through therapeutic angiogenesis have received great interest during last few years. The recent advances in enhancing the stability of mRNA through chemical modifications have introduced modified mRNA (mmRNA) as a potential therapeutic candidate for various applications including angiogenesis. Modi-fied mRNA delivery may represent a safer and more promising strategy of therapeutic angiogenesis compared to gene and protein delivery, by inducing the expression of angiogenic proteins through the cells’ translational machinery, without integrating into the genome. The aim of this study was to develop mmRNA transcript-in-corporated scaffolds as a controlled delivery system for angiogenic factors, for revascularization of ischemic skeletal muscles. The effect of physicochemical properties of the porous alginate scaffold on mmRNA release kinetics, transfection efficiency, and cell viability was studied. Taking advantage of electrostatic interactions

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between the negatively charged alginate scaffold and cationic lipid/mmRNA complexes, sustained release of GFP mmRNA was obtained for up to one week. The live/dead cell viability studies demonstrated high cell viability of human microvascular endothelial cells (HMEC-1) on alginate scaffold at a lower degree of cross-linking. The mmRNA transcript-incorporated scaffold can be potentially employed as a promising therapeutic approach for revascularization in broad range of ischemic diseases.

23. TBX5-Clover2/NKX2-5-TagRFP iPSCs for Simultaneously Isolating Human Lineage-Specific Cardiovascular Cells

Joe Z Zhang1,2,3, Vittavat Termglinchan1,2,3, Ilanit Itzhaki1,2,3, Ningyi Shao1,2,3, Vicky Y Wang4, Ning Ma1,2,3, Tomoya Kitani1,2,3, Alex C Y Chang1,5, Jaecheol Lee1,2,3, Chun Liu1,2,3, Nazish Sayed1,2,3, Haodi Wu1,2,3, Chi Keung Lam1,2,3, Helen M Blau1,5, Joseph C Wu1,2,3

1) Stanford Cardiovascular Institute; 2) Department of Medicine, Division of Cardiology; 3) Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, California, USA; 4) Auckland Bioengineering Institute, University of Auckland, Level 6, UniServices House, 70 Symonds Street, Auckland 1142, New Zealand; 5) Department of Microbiology and Immunology, Stanford University School of Medicine, California, USA

Heterogeneity and immaturity of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) significantly impede their applications in regenerative medicine, disease modelling, and personalized drug testing. Here, we developed a method for simultaneous isolation of lineage-specific ventricular-like, atrial-like and nodal-like CMs with high purity using a TBX5-Clover2/NKX2-5-TagRFP hiPSC reporter system. We also demonstrate that TBX5+/NKX2-5+ CMs possess more mature electrophysiological and metabolic phenotypes.

24. High Spatiotemporal Resolution Mapping of Cardiac Arrhythmic Activities in Porcine Model Using a Novel Elastic Polymeric Device

Xinyuan Zhang1, Jia Liu2, Yuxin Liu3, Zhenan Bao2, Anson Lee1

1) Department of Cardiothoracic Surgery, Stanford School of Medicine; 2) Department of Chemical Engineering; and 3) Department of Bioengineering, Stanford University

Atrial fibrillation (AF) is the most prevalent arrhythmia affecting 3-6 million U.S. individuals, and is a major contributor to morbidity and mortality. Recently, a fully stretchable microscale sensor array has been devel-oped, which provides intimate coupling and enables us to map cardiac arrhythmia activities with high spa-tiotemporal resolution. We have successfully demonstrated that the electrode array is capable of measuring epicardial cardiac arrhythmic activities, corresponding to simultaneous endocardial basket catheter mapping in an in vivo porcine model. This will enable us to better track and understand initiation and maintenance of atrial fibrillation.

25. Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells

Ming-Tao Zhao1,2,3,*, Ning-Yi Shao1,2,3*, Shijun Hu1,2,3*, Ning Ma1,2,3, Rajini Srinivasan4, Fereshteh Jahanbani5, Jaecheol Lee1,2,3, Sophia L. Zhang1,2,3, Michael P. Snyder5,**, Joseph C. Wu1,2,3,**

1) Stanford Cardiovascular Institute; 2) Department of Medicine, Division of Cardiology; 3) Institute of Stem Cell Biology and Regenerative Medicine; 4) Department of Chemical and Systems Biology; 5) Department of Genetics, Stanford University School of Medicine, Stanford, California 94305, USA. * Co-first authors; ** Co-corresponding authors

Rationale: Regulatory DNA elements in the human genome play important roles in determining the transcrip-tional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is a mystery how chromatin marks in regulatory DNA elements are modulated to establish

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cell type-specific gene expression in the human heart. Objective: We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. Methods and Results: We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (n=84) in human endothelial cells (ECs: CD31+CD144+), cardiac progenitor cells (CPCs: Sca1+), fibroblasts (FBs: DDR2+), and their respective induced pluripotent stem cells (iPSCs). We uncovered two classes of regulatory DNA elements: Class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas Class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both Class I and Class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, Class I promot-ers displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human iPSCs and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells, and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Conclusions: Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering the epigenetic modulation of regulatory DNA elements that fine-tune spatiotemporal gene expression in human cardiac development and diseases.

26. A Shape-controlled Tunable Microgel Cell Delivery Platform for Low-dose Delivery of Primed Stem Cells for In Vivo Therapeutic Neovascularization

D Thomas1, 2, 3, G Marsico2, A Thirumaran3, X Chen3, B Lukasz4, B Rodriguez4, M Marchetti-Deschmann5, T O’Brien1, 2, A Pandit1

1 Cardiovascular Institute, Stanford University2 CÚRAM, National University of Ireland Galway3 REMEDI, National University of Ireland Galway4 Conway Institute, University College Dublin5 Institute of Chemical Technologies and Analytics, TU Vienna

Hydrogel-based cell delivery platforms fabricated from biomaterials have evolved from a cytoprotective de-sign to a functional biomaterial design. During natural tissue regeneration, cellular microenvironment and sol-uble cues influence reparative mesenchymal stem cells (MSCs). Prolonged ischemia and lack of blood supply drive cellular apoptosis and tissue death. However, delivery of primed stem cells via a tunable microenviron-ment triggers an ‘angiocrine’ response, driving therapeutic angiogenesis1. In this study, it is hypothesised that delivery of primed human MSCs on a shape-controlled microgel platform at a low-cell dose promotes angio-genesis in a hindlimb ischemia (HLI) model. Optimized 2mg/ml collagen microgels were fabricated by dispens-ing type-I collagen with 4S-Star-PEG and hMSCs at 0.8x106 cell density on to a hydrophobic surface1. Balb/c nude mice underwent unilateral HLI and were divided into five groups (n=12/group); PBS; microgels alone; microgels with 50,000 hMSCs; 50,000 and 1,000,000 cells alone. Laser-Doppler perfusion and pathological markers of severity were assessed between the groups. Histological and molecular evaluation of inflammation and angiogenesis were assessed using immunohistochemistry, multiplex ELISA, gene expression arrays and and MALDI-imaging mass spectrometry. Statistical analysis was performed using one-way ANOVA with p<0.05. Perfusion analysis revealed higher perfusion in hMSC embedded microgels 60%±20% compared to controls. Immunohistochemistry revealed increased angiogenesis and reduction in inflammation at day 21. hMSC embedded microgels showed significant up-regulation of protein and gene markers related to angiogenesis, and changes in tissue N-glycan profile. Microgel delivery of primed hMSCs at a low-cell dose was demonstrated to promote functional angiogenesis in a severe murine model of HLI. Acknowledgement: Science Foundation Ireland (SFI) under grant no.09/SRC/B1794. Reference: 1. Thomas D. et al, Biomaterials, 35, 8757-66, 2014.

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cvi.stanford.edu